Description:

Objective:

The objective of this research project
is to develop a pretreatment process suitable for enzymatic conversion of agricultural
residues into fermentable sugars. The proposed process uses aqueous ammonia (a
non-polluting substance) as the pretreatment reagent. Use of ammonia offers significant
economic and environmental merits because it is recycled easily and leaves no
residual effect on the environment. The proposed pretreatment is a part of the
integral biomass-to-fuels process that does not generate net CO2 (a green energy
process). It is a pretreatment method of our own invention. When it is incorporated
into the current biomass saccharification processes, it can accomplish a near
complete fractionation of biomass into the three major constituents (cellulose,
hemicellulose, and lignin). We intend to expand the fundamental knowledge base
of this method and advance it to a point where it can be evaluated as a process
technology.

Progress Summary:

The first year of this project was focused
on fractionation of corn stover by treatments with aqueous ammonia. Utilization
efficiency of lignocellulosic biomass can be improved significantly by fractionation
of the biomass. A two-stage percolation process was investigated for pretreatment
and fractionation of corn stover. This process is composed of hot water treatment
followed by treatment with aqueous ammonia, both applied in a flow-through
(percolation) reactor. The first stage processing is intended for hemicellulose
removal, whereas the second stage is intended for delignification. Ammonia
is easily recoverable, and it poses no environmental problem in aqueous solution.
The treated end product was found to be very high in cellulose content and
also highly susceptible to enzymatic digestion. The conditions that achieve
satisfactory level of biomass fractionation and acceptable enzymatic hydrolysis
were identified in terms of reaction temperature, flow rate (retention time),
and reaction time for each stage. With proper operation of two-stage treatment,
fractionation of biomass was achieved to the extent that the xylan fraction
is hydrolyzed with 92 to 95 percent conversion and recovered with 83 to 86
percent yields; the lignin removal is 75 to 81 percent. The remaining solid
after two-stage treatment contained 78 to 85 percent cellulose. The two-stage
treatments enhanced the enzymatic digestibility to 90 to 96 percent with 60
filter paper units (FPU)/g of glucan, and 87 to 89 percent with 15 FPU/g of
glucan. The composition and digestibility data of the treated samples indicate
that the lignin content in the biomass is one of the major factors controlling
the enzymatic digestibility.

In the second phase of this work, a new pretreatment method, soaking in aqueous
ammonia (SAA), was investigated. In this method, a feedstock is soaked in aqueous
ammonia over an extended period (1-10 days) at room temperature. This is done
without agitation under atmospheric pressure. Treatment of corn stover by SAA
removes 55 to 74 percent of the lignin, but retains nearly 100 percent of the
glucan and 85 percent of the xylan. The SAA treatment of corn stover achieved
enzymatic digestibilities comparable to those of high temperature aqueous ammonia
treatments such as ammonia recycle percolation. The xylan remaining in the
corn stover after SAA was hydrolyzed, along with glucan, by cellulase enzyme
because of the presence of xylanase in “cellulase.” The SSA treated
corn stover was evaluated further by simultaneous saccharification and fermentation
(SSF) and by simultaneous saccharification and co-fermentation (SSCF). In the
standard SSF test using Saccharomyces cerevisiae (NREL-D5A), an ethanol yield
of 73 percent was obtained on the basis of the glucan content in the treated
corn stover. Xylose accumulation in the SSF appears to inhibit the cellulase
activity of glucan, limiting the yield of ethanol. In the SSCF test using recombinant
Escherichia coli KO11, both the glucan and xylose were utilized effectively,
giving an overall ethanol yield of 77 percent of the theoretical maximum, based
on glucan and xylan. With the SSCF results, the fact that the xylan fraction
is retained is a desirable feature in pretreatment because the overall bioconversion
can be carried out in a single step without separate recovery of xylose from
the pretreatment liquid.

To reduce the treatment time in the SAA, increase of treatment temperature
was attempted. In the modified SSA, corn stover was soaked in 15 to 30 weight
percent (wt %) aqueous ammonia at 40-90°C for 6 to 24 hours. The optimum
treatment conditions of this process were 15 wt % of NH3, 60°C, 1:6 solid-to-liquid
ratio, and 12 hours of treatment time. The modified SAA retained 85 percent
of the xylan and removed 62 percent of the lignin. Under optimum treatment
conditions, the enzymatic digestibility of glucan was enhanced from 17 to 85
percent for glucan with 15 FPU/g-glucan enzyme loading, and 78 percent of the
xylan in the treated biomass was hydrolyzed by cellulase enzyme. The SSCF test
of the modified SAA samples (3% weight/volume glucan loading, recombinant E.
coli KO11) has shown highly effective glucan and xylan utilization for eventual
conversion into ethanol. The overall ethanol yield of the SSCF was 77 percent
of the theoretical maximum, based on glucan and xylan. The maximum ethanol
concentration reached 19.2 g/L, and it occurred at 96 hours.

Future Activities:

In the second year of this project, we plan to investigate
the process aspects of the pretreatment method we developed in the first year.
This will involve development of a laboratory-scale continuous reactor that
can simulate the ammonia recycle percolation process.

Additionally, we plan to investigate the production of value-added chemicals
other than ethanol from the pretreated corn stover. The products under consideration
are xylooligosaccharides (high-value food additive) and lactic acid.

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.